The present invention relates to novel chemiluminescent acridinium compounds that have emission maxima close to or in the near infrared (NIR) region ( greater than 590 nm). The structural requirements for such long wavelength-emitting acridinium compounds are disclosed herein. These novel acridinium compounds when used in conjunction with short wavelength-emitting acridinium esters (with emission maxima below 450 nm) should be highly useful labels for the simultaneous detection of multiple target analytes in immunoassays or nucleic acid assays due to the extremely small or negligible spectral overlap between the different labels. A true and effective optical segregation of the two or more specific signals associated with different analytes can be easily achieved by the use of optical filters in conjunction with a simple algorithmic operation for minor correction of any low level of cross-talk. From practical standpoint, minimal spectral overlap is thus the key factor that is necessary for the simultaneous and accurate measurement of different analytes. A further application of the compounds described here is in situations where there is optical interference from biological samples at short wavelength (under 500 nm). Under these conditions, these novel acridinium compounds should be useful, alternative, labels, permitting the optical separation of non-label related luminescence from the specific, chemiluminescent signal generated front the binding complex. Finally, long-wavelength acridinium compounds used in conjunction with detectors such as CCD cameras offer the potential for improved assay sensitivity since these detectors are remarkably efficient in reading long wavelength signals.
Acridinium esters (AE) provide for an extremely sensitive method of detection and are useful chemiluminescent signal molecules that have been used extensively as labels in immunoassays and nucleic acid assays. U.S. Pat. Nos. 4,745,181; 4,918,192; 5,110,932 first described hydrolytically stable Polysubstituted Aryl Acridinium Esters (PAAE) which are useful for analytical measurements and became the first chemiluminescent acridinium compounds that enabled ligand binding assays to meet the stringent conditions required for commercialization owing to their remarkable stability. Subsequently, U.S. Pat. Nos. 5,241,070; 5,538,901; and 5,663074 described nucleophilic PAAE useful for the direct labeling of diverse organic molecules which lack nucleophilic functional groups. The utility of PAAE was further enhanced with the advent of Functionalized Hydrophilic PAAE (U.S. Pat. No. 5,656,426) which increased the quantum yield of PAAE and enhanced the performance of PAAE-labeled binding partners in terms of the observed signal to noise ratios and the sensitivities of various binding assays. This was primarily due to the introduction of hydrophilic group at the acridinium nucleus which increased the aqueous solubility of the compound and also unexpectedly increased the quantum yield of light production. Additionally, introduction of ionizable groups at the phenoxy moiety produced another sub-class of hydrophilic PAAE (U.S. Pat. Nos. 5,227,489; 5,449,556; and 5,595,875) which could be encapsulated in large numbers within biomolecule-functionalized, liposomes with extremely low leakage over prolonged storage. The last application further enhanced the utility of PAAE.
M. Kawaguichi, et al. (Bioluminescence and Chemiluminescence, Proceedings of 9th International Symposium 1996, Ed. Hastings, Kricka and Stanley, John Wiley and Sons, 1997, pp. 480-484) have described stabilized phenyl acridinium esters for chemiluminescent immunoassays. AE derivatives with additional methyl substitutions at C-1, which are optional at C-3 of the acridinium nucleus with matching mono- or di-methyl substitutions at the ortho-positions of the phenoxy moiety, were shown to have excellent stability in aqueous solution.
EP 0324,202 A1 and subsequently EP 0609,885 A1 both describe acridinium esters with functional groups substituted at the nitrogen atom of the acridinium nucleus. The latter application further describes alternate substituents such as the biphenyl or naphthylmoieties as possible replacements for the phenyl group. These types of acridinium compounds are reported to have emission maxima at 420 nm.
Mattingly, et al. (U.S. Pat. Nos. 5,468,646 and 5,543,524) describe chemiluminescent acridinium salts, their methods of preparation, their antibody conjugates, and their applications in immunoassays. These acridinium salts belong to another class of compounds termed acridinium sulfonylamides (or N-sulfonylacridinium carboxamides). The acridinium sulfonylamides (AS) have aqueous stabilities which are comparable with PAAE. No emission maxima were reported for the AS described therein. However, since the same acridone species should be generated from both classes of these compounds during their reaction with alkaline peroxide, the emission maxima for the acridinium sulfonylamides is expected to be in the blue region.
Mattingly, et al. further describe and claim the analogous chemiluminescent phenanthridinium salts, their methods of preparation, their antibody conjugates, and their applications in immunoassays, in U.S. Pat. Nos. 5,545,739; 5,565,570, and 5,669,819. Additionally, in these patents a general structure of acridinium sulfonylamides is described showing possible substitutents of a Markush group at the acridinium nucleus. No particular benefits about the substitutents were stated. None of the AS derivatives depicted by the general structure fits the teachings described by the present invention. Finally, the above patents do not describe any attempt to extend the wavelength of light emission of the acridinium sulfonylamides nor do they outline any strucuture-activity rationale about how this may be achieved.
Conventional acridinium compounds, such as those described in the aforementioned patents and literature, emit light with maxima at about 428 nm upon reaction with hydrogen peroxide in strong alkaline solution. Acridinium compounds which emit light of wavelength maxima  greater than 500 nm have also been described in the prior art. U.S. Pat. Nos. 5,395,752; 5,702,887 and 5,879,894 by Law et al. describe novel, long-emission acridinium esters (LEAE), where a fused, benzacridinium system is employed to extend the wavelength of emission of the acridinium ester. In the copending PCT application PCT/IB98/00831, Jiang et al. have further extended the PAAE emission maxima well into the region of 600-700 nm by utilizing the principle of energy transfer. This entailed the covalent coupling of luminophores to acridinium ester. Then the chemiluminescent reactions of these conjugates were initiated by treatment with alkaline peroxide, light emission was observed at long wavelengths where the wavelength maxima depended upon the structure of the luminophore.
EP 0 478 626 B1 and U.S. Pat. No. 5,656,207 by Batmanghelich et al. outline a structure for a purported, long-wavelength-emitting acridinium ester, in which an extended conjugation system is drawn by appending a substituted carboxybutadienyl group to the acridinium ester. However, in the Batmanghelich patents, neither the synthesis of this acridinium ester nor its emission properties was described to enable and substantiate the claim of light emission maxima of 500-700 nm, as already pointed out in the U.S. patent application Ser. No. 08/308,772, now U.S. Pat. No. 5,879,894.
Other non-acridinium ester-based, long emitting chemiluminescent compounds related to stable 1,2-dioxetanes have been described by Bronstein et. al. in U.S. Pat. No. 4,931,223. Said patent discloses chemiluminescent 1,2-dioxetanes comprised of enzyme-cleavable, functional groups and light emitting fluorophores with different emission wavelengths. Specific preferred embodiments include a acetoxybenzopyran-substituted stable dioxetane (A), a phosphoryloxy-benzopyran-substituted stable dioxetane (B), and a xcex2-galactosyloxy-benzothiazolyl-benzopyran-substituted stable dioxetane (C). The dioxetane A emits light with a 450 nm wavelength maximum when its acetoxy group is cleaved by an esterase. The dioxetane B emits lights at a 480 nm wavelength maximum when its phosphoryloxy group is cleaved by a phosphatase, while the dioxetane C emits light at 515 nm wavelength maximum upon treatment with the enzyme xcex2-galactosidase. The patent provides an example of a three-channel analysis for the simultaneous detection of HSV, CMV, and HPV in a nucleic acid probe simultaneous assay, using three, narrow band-pass optical filters to sort out the different color emissions from the aforementioned dioxetanes. The levels of HSV, CMV, and HPV present in the sample were correlated by the corresponding image brightness. Because the three light emission maxima are so close together, most of the spread from each emission spectrum had to be cut off by the narrow band pass filters to remove signal from the overlapping regions. This resulted in a very little usable amount of signal for each assay component and greatly limited the assay sensitivity and perhaps accuracy.
Edwards et al. [J. Biolumin. and Chemilumin., 5, 1 (1990)] have reported an another chemiluminescent dioxetane, 3-(2xe2x80x2-spiroadamantane)-4-methoxy-4-(7xe2x80x3-acetoxy)naphth-2xe2x80x2-yl-1,2-dioxetane, which emits green light (maximum of 550 nm) with a bathochromic shift of 90 nm in comparison to its 6xe2x80x3-acetoxy substituted isomer. This is attributed to the different position of the enzymatically cleavable acetoxy substituent that gives rise to an oxide anion substituent responsible for triggering dioxetane decomposition. Similar application of the two isomeric dioxetane compounds for the simultaneous detection of several analytes was suggested in the paper.
In this invention, we describe the design and synthesis of novel acridinium compounds which emit light with wavelength maxima  greater than 590 nm upon reaction with hydrogen peroxide. These acridinium compounds contain some key structural features which are critical for observing long-wavelength emission. These results along with our earlier observations described in U.S. Pat. No. 5,395,752 provide sound and experimentally verified rules for the design and synthesis of long-wavelength-emitting acridinium compounds.
For the improved measurement of the NIR chemiluminescent signal we also disclose in the present invention a modified, semi-automatic luminescence analyzer in which the red-insensitive photomultiplier tube has been replaced with a state-of-the-art low-noise, cooled CCD detector utilized in photon counting mode. By comparing the quantitated signals obtained from the original and modified analyzers we demonstrated an improvement in the specific activity of a NIR acridinium compound by about 40-fold. The use of a cooled CCD camera system for imaging chemiluminescent signal in the short wavelength region generated from a 1,2-dioxetane compound, was described by Martin, et. al. in J. Biolumin. Chemilumin. 9 (3), 145, 1994. The applications that were said to have been adapted to this imaging method, included various nucleic acid and immuno blottings, ELISA methods and DNA sequencing systems.
This invention identifies two sets of necessary and sufficient criteria for observing long-wavelength emission from acridinium compounds:
Set A:
(a) The creation of an extended conjugation system by the attachment of appropriate functional groups on the acridinium nucleus (electronic requirement).
(b) Coplanarity of the attached functional group and the acridone moiety during light emission (geometry requirement).
(c) Said functional group must consist of at least one aromatic ring and one electron-donating atom or group with an extra pair of electrons which can readily delocalize into the extended xcfx80 system to which the heteroatom is directly attached or built into, and establish stable extended resonance with the electron-withdrawing carbonyl moiety of the light emitting acridone. Such electron-donating atom or group that exists in the form of an anion has particularly strong effect to further the bathochromic shift of the emission wavelength.
Set B:
(a) A direct attachment at one or more of positions C-2, C-4, C-5, or C-7 of the acridinium nucleus, of electron-donating atoms or groups having extra pair(s) of electrons. The electron-donating entities can be the same or different if more than one electron-donating entity is used. Such electron-donating atom or group that exists in the form of an anion has particularly strong effect to further the bathochromic shift of the emission wavelength.
For molecules for which the above criteria are met such as LEAE, 3-HS-DMAE, and 2-hydroxy-DMAE long wavelength-emission exceeding 500 nm and reaching into NIR region is expected and observed.
Preferably, the utility of an NIR-AC of comparable quantum yield as the conventional acridinium compounds goes hand-in-hand with the employment of a luminenscence detector of good to excellent detection efficiency. To achieve efficient NIR signal detection and facilitate the performing of diagnostic assays, a further objective of the present invention is the advance of a concept and the realization of substituting a state-of-the-art charge-coupled device (CCD) detector for the red-insensitive photomultiplier tube (PMT) in a conventional fully or semi-automatic analyzer such as MLA-II of Chiron Diagnostics, Walpole, Mass.